Plant VDE genes and methods related thereto

ABSTRACT

DNA sequences encoding plant vde enzymes are provided herein. The sequences may be joined to heterologous DNA sequences for use as probes and in DNA constructs to modify the genotype of a host organism. DNA constructs and methods are provided to modify a host cell phenotype by altering the amount of photoprotection enzyme present in the host cell. In plastid containing host cells, zeaxanthin levels and sensitivity to light can be modified through alterations in the level of vde enzymes.

This application claims benefit of provisional application No.60,023,502, filed Aug. 6, 1996, which is a provisional of 60,006,315,filed Nov. 7, 1995.

FIELD OF THE INVENTION

This invention relates to genes encoding plant violaxanthin de-epoxidase(vde) and methods of use related to the protein and the nucleic acidsequences. The invention is exemplified by methods of causing increasedexpression or decreased expression of plant vde genes in plants.Included are plants produced by the method.

INTRODUCTION Background

Plant carotenoids are found in the membranes of chloroplasts andchromoplasts. They are instrumental in the photoprotective mechanisms ofplants. Also, plant carotenoids have significant dietary implications.Thus, from an agronomic as well as a nutritional standpoint, study ofthe plant carotenoids and the enzymes involved in the biosynthesis ofcarotenoids is of interest.

Of particular interest are the late stages of the carotenoidbiosynthetic pathway in plants, the xanthophyll cycle and its importancein photoregulation of photosynthesis. Photosynthesis is the process thatenable plants to use light energy for growth and development. Thus, theavailability of light of appropriate quality and quantity(photosynthetically active radiation or "PAR") is critical for plantgrowth and development. Ironically, light can also damage plants becauseplants have limited capacity to use light. When light intensity exceedsthis capacity, irreversible damage can occur.

Plants have developed various mechanisms to cope with excess light suchas varying leaf orientation or developing reflective surfaces. Suchmechanisms appear to be specialized phenotypic strategies that arelimited to certain types of plants. One mechanism that is apparentlyused by all plants examined so far is the dissipation of excess energyas heat in the antenna (light absorbing structures) of thephotosynthetic apparatus. Most of the excess energy is discarded as heatby a complex feed-back regulatory system that involves thetransthylakoid ΔpH and formation of antheraxanthin and zeaxanthincatalyzed by violaxanthin de-epoxidase (vde) in the xanthophyll cycle.This system, termed energy dependent non-radiative energy dissipation ornon-photochemical fluorescence quenching, reduces the quantum efficiencyof photosystem II (PSII), helping to prevent PSII over reduction andphotoinhibitory damage. In effect, this system provides a means to dumpexcess energy before it can damage the photosynthetic apparatus. Thesystem has a wide dynamic range, both qualitatively and quantitatively,which enables it to function effectively over a wide-range ofenvironmental conditions.

The ability to manipulate aspects of the xanthophyll cycle throughgenetic engineering techniques would permit the rapid introduction ofimproved plant varieties. However, it has been difficult to obtainpurified fractions of the enzymes involved in the pathway and, prior tothis invention, the corresponding genes have not been cloned.

SUMMARY OF THE INVENTION

DNA sequences encoding plant vde enzymes are provided herein. Thesequences may be joined to heterologous DNA sequences for use as probesand in DNA constructs to modify the genotype of a host organism. DNAconstructs and methods are provided to modify a host cell phenotype byaltering the amount of photoprotection enzyme present in the host cell.In plastid containing host cells, zeaxanthin levels and sensitivity tolight can be modified through alterations in the level of vde enzymes.

For example, over expression of vde is expected to increase thetolerance of plants to high light, drought and temperature stress(stress conditions exacerbate the condition of excess light). Also,plants that are not currently tolerant to high light or low temperaturesare expected to become more tolerant to these stresses. Plants that arebetter adapted to light stress are expected to be more productive and/ormore resistant to disease. Alternatively, the under expression, orinhibition of vde activity is expected to increase photosyntheticefficiency under low light. The growing range of plants, crops, treesand ornamentals, could thus be modified.

Specific plant vde's are described. In particular, a 55 kD lettuce vdehaving the cDNA sequence and deduced amino acid sequence as shown inFIG. 1, a tobacco vde having the cDNA sequence and deduced amino acidsequence as shown in FIG. 2, and an Arabidopsis vde having the cDNAsequence and deduced amino acid sequence as shown in FIG. 3, aredescribed. FIG. 4 provides a comparison at the amino acid level of theproteins of FIGS. 1-3. In this amino acid sequence comparison the trasitpeptides for the three sequences are boxed. Identical amino acids aredenoted by a hyphen. Gaps inserted to optimize sequence alignments aredenoted with a period. The thirteen highly conserved cysteine residuesare denoted with an asterisk.

FIG. 5 is a comparison of the identity and similarity of pre-protein andmature protein vde. As can be seen from FIG. 5, diverse vde's havesequences with about 75% sequence identity with one another at the aminoacid level. Thus, vde sequences having at least about 75% homology toamino acid sequences in FIG. 1, FIG. 2 or FIG. 3 are also contemplatedhereunder.

Nucleic acid sequences encoding a plant vde having at least about 60%sequence identity, and more preferably at least about 70% sequenceidentity, with the sequences in FIGS. 1, 2 or 3, and are likewisecontemplated herein. For instance, a comparison of tobacco and lettucevde nucleic acid sequences give 76% identity, excluding the transitpeptides. A high degree of sequence identity at the N-terminus isparticularly preferred. Other related plant photoregulatory sequenceshaving high degrees of similarity with fragments of the vde sequencesshown are also contemplated.

In a different aspect of this invention, nucleic acid sequences relatedto the exemplified lettuce, tobacco and arabidopsis vde sequences ofthis invention are described with details regarding methods to obtainsuch sequences from a variety of sources and their use. In addition,cDNA sequences encoding mature vde's are given as well as transitpeptides, mRNA, genomic plant vdes, and plant vde regulatory regions.

In a further aspect of this invention, methods of producing vde in hostcells are described. In plastid containing cells, modifications in thexanthophyll cycle, particularly in the ratio of violaxanthin as tozeaxanthin are contemplated via increased production of vde or decreasedproduction of vde. This will have applications in the increased feedvalue of plants. Zeaxanthin levels are important to crops such asalfalfa whose value in part is due to xanthophyll content.

Results from studies of transgenic plants demonstrates thatxanthophyll-mediated energy dissipation in LHCII apparently protectsPSII against the potentially damaging effects of high light. Thisprotection is induced by the combined effects of a thylakoid ΔpH and thepresence of zeaxanthin and antheraxanthin formed by violaxanthinde-epoxidase (vde) activity.

DESCRIPTION OF THE FIGURES

FIG. 1 cDNA sequence (SEQ ID NO: 1) for romaine lettuce vde and deducedpolypeptide sequence. The underlined sequences are those determined frompeptide sequencing of purified lettuce vde. The polypeptide sequencebegins at the first methionine of the open reading frame and is precededby three termination codons in the same reading frame.

FIG. 2 cDNA sequence (SEQ ID NO: 2) for tobacco (Nicotiana tabacum cv.Xanthi) vde and deduced polypeptide sequence.

FIG. 3 cDNA sequence (SEQ ID NO: 2) for Arabidopsis thaliana (var.columbia) vde and deduced polypeptide sequence.

FIG. 4 provides a comparison of the amino acid sequences of the proteinsof FIGS. 1-3.

FIG. 5 shows the percent similarity between the the proteins of FIGS.1-3.

FIG. 6 provides a comparison of hyropathy profiles for the vdes of threespecies.

FIG. 7 provides a time-course comparison of effects of expressed vde.

FIG. 8 is a table showing the results of pigment analysis of leaves ofcontrol and 18 vde-antisense tobacco plants (TAS-#).

FIG. 9 shows the results of a control plant extraction for vde.

FIG. 10 shows the results of extraction for vde in an antisense vdeplant.

DETAILED DESCRIPTION OF THE INVENTION

A plant violaxanthin de-epoxidase or "vde" of this invention includesany sequence of amino acids, such as a protein, polypeptide or peptide,obtainable from a plant source, which demonstrates the ability tocatalyze the production of zeaxanthin from violaxanthin under plantenzyme reactive conditions. By "enzyme reactive conditions" is meantthat any necessary conditions that are available in an environment(i.e., such factors as temperature, pH, lack of inhibiting substances)which will permit the enzyme to function.

By "plant" is meant any plastid-containing organism. A "higher plant"shall mean any differentiated, multi-cellular plastid-containingorganism. Of particular interest are plant vde's from angiosperms, bothdicotyledonous and monocotyledonous plants.

In this invention, the cDNA sequence of a lettuce (FIG. 1), tobacco(FIG. 2) and Arabidopsis (FIG. 3) vde gene are provided. Transit peptideregions are identified in FIG. 4. From these sequences, genomicsequences may be obtained and the corresponding transcriptional andtranslational regulatory regions determined. Also, using the lettuceand/or tobacco sequences provided, vde genes from other sources may beobtained. In particular, it is found that the N-terminal regions of thelettuce, tobacco, Arabidopsis and spinach proteins are conserved andtherefore, an N-terminal peptide such as "VDALKTCACLLK" will findparticular use in obtaining related sequences.

Constructs for use in the methods may include several forms, dependingupon the intended use of the construct. Thus, the constructs includevectors, transcriptional cassettes, expression cassettes and plasmids.The transcriptional and translational initiation region (also sometimesreferred to as a "promoter") preferably comprises a transcriptionalinitiation regulatory region and a translational initiation regulatoryregion of untranslated 5' sequences, "ribosome" binding sites,"responsible for binding mRNA to ribosomes and translational initiation.It is preferred that all of the transcriptional and translationalfunctional elements of the initiation control region are derived from orobtainable from the same gene. In some embodiments, the promoter will bemodified by the addition of sequences, such as enhancers, or deletionsof nonessential and/or undesired sequences. By "obtainable" is intendeda promoter having a DNA sequence sufficiently similar to that of anative promoter to provide for the desired specificity of transcriptionof a DNA sequence of interest. It includes natural and syntheticsequences as well as sequences which may be a combination of syntheticand natural sequences.

A transcriptional cassette for transcription of a nucleotide sequence ofinterest will include in the direction of transcription, a transcriptioninitiation region and optionally a translational initiation region, aDNA sequence of interest, and a transcriptional and optionallytranslational termination region functional in the host cell ofinterest. When the cassette provides for the transcription andtranslation of a DNA sequence it is considered an expression cassette.One or more introns may also be present. Other sequences may also bepresent, including those encoding transit peptides.

The use of amino acid sequences from vde peptides to obtain nucleic acidsequences which encode lettuce vde is described herein. For example,synthetic oligonucleotides are prepared which correspond to the vdepeptide sequences. The oligonucleotides are used as primers inpolymerase chain reaction (PCR) techniques to obtain partial DNAsequence of vde genes. The partial sequences so obtained are then usedas probes to obtain vde clones from a gene library prepared from lettucetissue. Alternatively, where oligonucleotides of low degeneracy can beprepared from particular vde peptides, such probes may be used directlyto screen gene libraries for vde gene sequences. In particular,screening of cDNA libraries in phage vectors is useful in such methodsdue to lower levels of background hybridization.

A nucleic acid sequence of a plant vde of this invention may be a DNA orRNA sequence, derived from genomic DNA, cDNA, mRNA, or may besynthesized in whole or in part. The gene sequences may be cloned, forexample, by isolating genomic DNA from an appropriate source, andamplifying and cloning the sequence of interest using a polymerase chainreaction (PCR). Alternatively, the gene sequences may be synthesized,either completely or in part, especially where it is desirable toprovide plant-preferred sequences. Thus, all or a portion of the desiredstructural gene (that portion of the gene which encodes the vde protein)may be synthesized using codons preferred by a selected host.Host-preferred codons may be determined, for example, from the codonsused most frequently in the proteins expressed in a desired hostspecies.

One skilled in the art will readily recognize that antibodypreparations, nucleic acid probes (DNA and RNA) and the like may beprepared and used to screen and recover "homologous" or "related" vde'sfrom a variety of plant sources. Homologous sequences are found whenthere is an identity of sequence, which may be determined uponcomparison of sequence information, nucleic acid or amino acid, orthrough hybridization reactions between a known vde and a candidatesource. Conservative changes, such as Glu/Asp, Val/Ile, Ser/Thr, Arg/Lysand Gln/Asn may also be considered in determining sequence homology.Amino acid sequences are considered homologous by as little as 25%sequence identity between the two complete mature proteins. (Seegenerally, Doolittle, R. F., OF URFS and ORFS (University Science Books,Calif., 1986.)

Thus, other plant vde's may be obtained from the specific exemplifiedlettuce, tobacco and Arabidopsis sequences provided herein. Furthermore,it will be apparent that one can obtain natural and synthetic plantvde's, including modified amino acid sequences and starting materialsfor synthetic-protein modeling from the exemplified plant vde's and fromplant vde's which are obtained through the use of such exemplifiedsequences. Modified amino acid sequences include sequences which havebeen mutated, truncated, increased and the like, whether such sequenceswere partially or wholly synthesized. Sequences which are actuallypurified from plant preparations or are identical or encode identicalproteins thereto, regardless of the method used to obtain the protein orsequence, are equally considered naturally derived.

Typically, a plant vde sequence obtainable from the use of nucleic acidprobes will show 60-70% sequence identity between the target vdesequence and the encoding sequence used as a probe. However, lengthysequences with as little as 50-60% sequence identity may also beobtained. The nucleic acid probes may be a lengthy fragment of thenucleic acid sequence, or may also be a shorter, oligonucleotide probe.When longer nucleic acid fragments are employed as probes (greater thanabout 100 bp), one may screen at lower stringencies in order to obtainsequences from the target sample which have 20-50% deviation (i.e.,50-80% sequence homology) from the sequences used as probe.Oligonucleotide probes can be considerably shorter than the entirenucleic acid sequence encoding a vde enzyme, but should be at leastabout 10, preferably at least about 15, and more preferably at leastabout 20 nucleotides. A higher degree of sequence identity is desiredwhen shorter regions are used as opposed to longer regions. It may thusbe desirable to identify regions of highly conserved amino acid sequenceto design oligonucleotide probes for detecting and recovering otherrelated vde genes. Shorter probes are often particularly useful forpolymerase chain reactions (PCR), especially when highly conservedsequences can be identified. (See, Gould, et al., PNAS USA (1989)86:1934-1938.)

To determine if a related gene may be isolated by hybridization with agiven sequence, the sequence is labeled to allow detection, typicallyusing radioactivity, although other methods are available. The labeledprobe is added to a hybridization solution, and incubated with filterscontaining the desired nucleic acids, either Northern or Southern blots(to screen desired sources for homology), or the filters containing cDNAor genomic clones to be screened. Hybridization and washing conditionsmay be varied to optimize the hybridization of the probe to thesequences of interest. Lower temperatures and higher salt concentrationsallow for hybridization of more distantly related sequences (lowstringency). If background hybridization is a problem under lowstringency conditions, the temperature can be raised either in thehybridization or washing steps and/or salt content lowered to improvedetection of the specific hybridizing sequence. Hybridization andwashing temperatures can be adjusted based on the estimated meltingtemperature of the probe as discussed in Beltz, et al. (Methods inEnzymology (1983) 100:266-285).

A useful probe and appropriate hybridization and washing conditionshaving been identified as described above; cDNA or genomic libraries arescreened using the labeled sequences and optimized conditions. Thelibraries are first plated onto a solid agar medium, and the DNA liftedto an appropriate membrane, usually nitrocellulose or nylon filters.These filters are then hybridized with the labeled probe and washed asdiscussed above to identify clones containing the related sequences.When a genomic library is used, one or more sequences may be identifiedproviding both the coding region and the transcriptional regulatoryelements of the vde gene from such plant source.

For immunological screening, antibodies to the vde protein can beprepared by injecting rabbits or mice with the protein purified from theoriginal plant source or expressed from a host cell, such methods ofpreparing antibodies being well known to those in the art. Eithermonoclonal or polyclonal antibodies can be produced, although typicallypolyclonal antibodies are more useful for gene isolation. Westernanalysis may be conducted to determine that a related protein is presentin a crude extract of the desired plant species, as determined bycross-reaction with the antibodies to the vde. When cross-reactivity isobserved, genes encoding the related proteins are isolated by screeningexpression libraries representing the desired plant species. Expressionlibraries can be constructed in a variety of commercially availablevectors, including lambda gt11, as described in Maniatis, et al.(supra).

All plants studied to date utilize the xanthophyll cycle, and thus anygiven plant species can be considered as a source of additional vdeproteins.

The nucleic acid sequences associated with plant vde proteins will findmany uses. For example, recombinant constructs can be prepared which canbe used as probes or will provide for expression of the vde protein inhost cells to produce a ready source of the enzyme. Other usefulapplications may be found when the host cell is a plant host cell,either in vitro or in vivo. For example, by increasing the amount of arespective vde available to the plant xanthophyll cycle, an increasedpercentage of zeaxanthin may be obtained. In a like manner, for someapplications it may be desired to decrease the amount of vdeendogenously expressed in a plant cell by anti-sense or some otherreducing technology such as co-supression. For example, to improvephotosynthetic efficiency of a plant under low light, decreasedexpression of a vde may be desired.

Thus, depending upon the intended use, the constructs may contain thesequence which encodes the entire vde protein, or a portion thereof. Forexample, where antisense inhibition of a given vde protein is desired,the entire vde sequence is not required. Furthermore, where vdeconstructs are intended for use as probes, it may be advantageous toprepare constructs containing only a particular portion of an vdeencoding sequence, for example a sequence which is discovered to encodea highly conserved vde region.

As discussed above, nucleic acid sequence encoding a plant vde of thisinvention may include genomic, cDNA or mRNA sequence. By "encoding" ismeant that the sequence corresponds to a particular amino acid sequenceeither in a sense or anti-sense orientation. By "extrachromosomal" ismeant that the sequence is outside of the plant genome of which it isnaturally associated. By "recombinant" is meant that the sequencecontains a genetically engineered modification through manipulation viamutagenesis, restriction enzymes, and the like.

A cDNA sequence may or may not contain pre-processing sequences, such astransit peptide sequences or targeting sequences to facilitate deliveryof the vde protein to a given organelle or membrane location. The use ofany such precursor vde DNA sequences is preferred for uses in plant cellexpression. A genomic vde sequence may contain the transcription andtranslation initiation regions, introns, and/or transcript terminationregions of the plant vde, which sequences may be used in a variety ofDNA constructs, with or without the vde structural gene. Thus, nucleicacid sequences corresponding to the plant vde of this invention may alsoprovide signal sequences useful to direct protein delivery into aparticular organelle or membrane location, 5' upstream non-codingregulatory regions (promoters) having useful tissue and timing profiles,3' downstream non-coding regulatory region useful as transcriptional andtranslational regulatory regions and may lend insight into otherfeatures of the gene.

Once the desired plant vde nucleic acid sequence is obtained, it may bemanipulated in a variety of ways. Where the sequence involves non-codingflanking regions, the flanking regions may be subjected to resection,mutagenesis, etc. Thus, transitions, transversions, deletions, andinsertions may be performed on the naturally occurring sequence. Inaddition, all or part of the sequence may be synthesized. In thestructural gene, one or more codons may be modified to provide for amodified amino acid sequence, or one or more codon mutations may beintroduced to provide for a convenient restriction site or other purposeinvolved with construction or expression. The structural gene may befurther modified by employing synthetic adapters, linkers to introduceone or more convenient restriction sites, or the like.

The nucleic acid or amino acid sequences encoding a plant vde of thisinvention may be combined with other non-native, or "heterologous",sequences in a variety of ways. By "heterologous" sequences is meant anysequence which is not naturally found joined to the plant vde,including, for example, combinations of nucleic acid sequences from thesame plant which are not naturally found joined together.

The DNA sequence encoding a plant vde of this invention may be employedin conjunction with all or part of the gene sequences normallyassociated with the vde. In its component parts, a DNA sequence encodingvde is combined in a DNA construct having, in the 5' to 3' direction oftranscription, a transcription initiation control region capable ofpromoting transcription and translation in a host cell, the DNA sequenceencoding plant vde and a transcription and translation terminationregion.

Potential host cells include both prokaryotic and eukaryotic cells. Ahost cell may be unicellular or found in a multicellular differentiatedor undifferentiated organism depending upon the intended use. Cells ofthis invention may be distinguished by having a plant vde foreign to thewild-type cell present therein, for example, by having a recombinantnucleic acid construct encoding a plant vde therein.

Depending upon the host, the regulatory regions will vary, includingregions from viral, plasmid or chromosomal genes, or the like. Forexpression in prokaryotic or eukaryotic microorganisms, particularlyunicellular hosts, a wide variety of constitutive or regulatablepromoters may be employed. Expression in a microorganism can provide aready source of the plant enzyme. Among transcriptional initiationregions which have been described are regions from bacterial and yeasthosts, such as E. coli, B. subtilis, Sacchromyces cerevisiae, includinggenes such as beta-galactosidase, T7 polymerase, tryptophan E and thelike.

For the most part, the constructs will involve regulatory regionsfunctional in plants. The open reading frame, coding for the plant vdeor functional fragment thereof will be joined at its 5' end to atranscription initiation regulatory region such as the wild-typesequence naturally found 5' upstream to the vde structural gene.Numerous other transcription initiation regions are available whichprovide for a wide variety of constitutive or regulatable, e.g.,inducible, transcription of the structural gene functions. Constitutivepromoters such as the CaMV 35S promoter, double 35S promoter, 34Sfigwort promoter may be useful. Promoters which express in plastidcontaining cells will be of special interest. Some such promoters arepreferentially expressed in plastid containing tissues, such as the ssupromoter. The transcription/translation initiation regions correspondingto such structural genes are found immediately 5' upstream to therespective start codons. In embodiments wherein the expression of thevde protein is desired in a plant host, the use of all or part of thecomplete plant vde gene is desired; namely all or part of the 5'upstream non-coding regions (promoter) together with the structural genesequence and 3' downstream non-coding regions may be employed. If adifferent promoter is desired, such as a promoter native to the planthost of interest or a modified promoter, i.e., having transcriptioninitiation regions derived from one gene source and translationinitiation regions derived from a different gene source, including thesequence encoding the plant vde of interest, or enhanced promoters, suchas double 35S CaMV promoters, the sequences may be joined together usingstandard techniques.

Expression of the vde transcript was followed in market romaine lettuceleaves that were dark adapted for an undetermined period of time. Thesame level of transcript was detected in both yellow leaves and rapidlyexpanding green leaves. However, a greater transcript level was detectedin mature green leaves. Two hybridizing transcripts were observed foreach sample indicating the possibility that the upper larger transcriptmay be processed to the slightly smaller transcript (1.7 kb) having thegreater level of hybridization. The increased level of transcript inmature green leaves of lettuce may be due to two possible reasons:higher expression occurs in tissues with a higher density of fullydeveloped chloroplasts or expression may be regulated by light intensitysince the mature green leaves receive a higher intensity of light thanthe immature leaves which are partially shielded in the center of thehead of lettuce. Hence, use of the vde promoter may be particularlyuseful in the transcription of vde nucleic acid sequences or for theexpression of other nucleic acid sequences of interest.

Regulatory transcript termination regions may be provided in DNAconstructs of this invention as well. Transcript termination regions maybe provided by the DNA sequence encoding the plant vde or a convenienttranscription termination region derived from a different gene source,for example, the transcript termination region which is naturallyassociated with the transcript initiation region. Where the transcripttermination region is from a different gene source, it will contain atleast about 0.5 kb, preferably about 1-3 kb of sequence 3' to thestructural gene from which the termination region is derived.

Plant expression or transcription constructs having a plant vde as theDNA sequence of interest for increased or decreased expression thereofmay be employed with a wide variety of plant life, particularly, plantlife where light regulation or zeaxanthin levels are important. Plantsof interest include, but are not limited to ornamental plant varieties,field and forage crops, including alfalfa and trees. Depending on themethod for introducing the recombinant constructs into the host cell,other DNA sequences may be required. Importantly, this invention isapplicable to dicot and monocot species alike and will be readilyapplicable to new and/or improved transformation and regulationtechniques.

The method of transformation in obtaining such transgenic plants is notcritical to the instant invention, and various methods of planttransformation are currently available. Furthermore, as newer methodsbecome available to transform crops, they may also be directly appliedhereunder. For example, many plant species naturally susceptible toAgrobacterium infection may be successfully transformed via tripartiteor binary vector methods of Agrobacterium mediated transformation. Inmany instances, it will be desirable to have the construct bordered onone or both sides by T-DNA, particularly having the left and rightborders, more particularly the right border. This is particularly usefulwhen the construct uses A. tumefaciens or A. rhizogenes as a mode fortransformation, although the T-DNA borders may find use with other modesof transformation. In addition, techniques of microinjection, DNAparticle bombardment, and electroporation have been developed whichallow for the transformation of various monocot and dicot plant species.

Normally, included with the DNA construct will be a structural genehaving the necessary regulatory regions for expression in a host andproviding for selection of transformant cells. The gene may provide forresistance to a cytotoxic agent, e.g. antibiotic, heavy metal, toxin,etc., complementation providing prototrophy to an auxotrophic host,viral immunity or the like. Depending upon the number of different hostspecies the expression construct or components thereof are introduced,one or more markers may be employed, where different conditions forselection are used for the different hosts.

Where Agrobacterium is used for plant cell transformation, a vector maybe used which may be introduced into the Agrobacterium host forhomologous recombination with T-DNA or the Ti- or Ri-plasmid present inthe Agrobacterium host. The Ti- or Ri-plasmid containing the T-DNA forrecombination may be armed (capable of causing gall formation) ordisarmed (incapable of causing gall formation), the latter beingpermissible, so long as the vir genes are present in the transformedAgrobacterium host. The armed plasmid can give a mixture of normal plantcells and gall.

In some instances where Agrobacterium is used as the vehicle fortransforming host plant cells, the expression or transcription constructbordered by the T-DNA border region(s) will be inserted into a broadhost range vector capable of replication in E. coli and Agrobacterium,there being broad host range vectors described in the literature.Commonly used is pRK2 or derivatives thereof. See, for example, Ditta,et al., (Proc. Nat. Acad. Sci., U.S.A. (1980) 77:7347-7351) and EPA 0120 515, which are incorporated herein by reference. Alternatively, onemay insert the sequences to be expressed in plant cells into a vectorcontaining separate replication sequences, one of which stabilizes thevector in E. coli, and the other in Agrobacterium. See, for example,McBride and Summerfelt (Plant Mol. Biol. (1990) 14:269-276), wherein thepRiHRI (Jouanin, et al., Mol. Gen. Genet. (1985) 201:370-374) origin ofreplication is utilized and provides for added stability of the plantexpression vectors in host Agrobacterium cells.

Included with the expression construct and the T-DNA will be one or moremarkers, which allow for selection of transformed Agrobacterium andtransformed plant cells. A number of markers have been developed for usewith plant cells, such as resistance to chloramphenicol, kanamycin, theaminoglycoside G418, hygromycin, or the like. The particular markeremployed is not essential to this invention, one or another marker beingpreferred depending on the particular host and the manner ofconstruction.

For transformation of plant cells using Agrobacterium, explants may becombined and incubated with the transformed Agrobacterium for sufficienttime for transformation, the bacteria killed, and the plant cellscultured in an appropriate selective medium. Once callus forms, shootformation can be encouraged by employing the appropriate plant hormonesin accordance with known methods and the shoots transferred to rootingmedium for regeneration of plants. The plants may then be grown to seedand the seed used to establish repetitive generations.

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are included forpurposes of illustration only and are not intended to limit the presentinvention.

EXAMPLES Example 1--Lettuce vde cDNA

Vde was purified from romaine lettuce (Lactuca sativa L. cv Romaine)chloroplasts and peptides from a tryptic digest along with theN-terminus were sequenced (Rockholm, Plant Physiol. (1996) 110:697-703).Two peptides (N-terminus and tryptic fragment #15, shown in FIG. 1) wereused to develop the oligonucleotides

5'-GAYGCHYTBAAGACHTGYGC-3' (216-fold degeneracy) and

5'TTGVARRTTDGGRATRAT-3' (144-fold degeneracy).

The partial cDNA for vde was amplified by 35 cycles of polymerase chainreaction (PCR) containing 25 pmol of each primer and lettuce cDNA usingan annealing temperature of 50° C. The PCR product was subcloned intopGEM-7Zf (Promega) by blunt-end cloning and sequenced. A cDNA librarywas constructed from poly(A)+ RNA isolated from a pooled sample ofvarious age romaine lettuce leaves using the Timesaver cDNA SynthesisKit (Pharmacia) and ligated into lambda-ZAPII (Stratagene). A total of2.5×10⁵ recombinant plaques were screened with the PCR product labeledby random priming and positive clones were plaque purified followed byin vivo excision of the plasmid. The cDNAs were subcloned into the Notlsite of pGEM-5Zf and both strands of cDNA were sequenced completelyusing an Applied Biosystems Model 373A automated sequencer. The Genbankaccession number is U31462.

The vde cDNA encompasses an open reading frame encoding a 473 amino acidprotein with a calculated Mr of 54,447. The deduced protein contains an125 amino acid putative transit peptide for transport into thechloroplast lumen where the enzyme is localized (Hager, Planta(1969)89:224-243). This was verified by in vitrotranscription/translation of two vde (vde1:-234 to 1526 bp and vde2:-65to 1578 bp of FIG. 1) cDNAs which produced a 55 Kd product on a sodiumdodecyl sulfate (SDS)-polyacrylamide gel. The N-terminus of the maturevde protein (amino acid #126) was determined by N-terminal sequencing ofpurified vde from romaine lettuce. Therefore, mature vde consists of a348 amino acid protein with a calculated Mr of 39,929 and a calculatedpI of 4.57.

The primary structure of the deduced mature vde exhibits somecharacteristic features. The protein is hydrophilic overall with 57.2%of the total amino acid residues having polar side chains. Threeinteresting domains were identified in the deduced mature vde includinga cysteine rich domain, a lipocalin signature and a highly chargeddomain. In the first domain 11 of the 13 total cysteines in the maturevde are present suggesting that this is most likely the site wheredithiothreitol (DTT), a known inhibitor of vde, has its effect. Thecysteines probably form more than one disulfide linkage since partialinhibition of vde activity with DTT results in an accumulation ofantheraxanthin. The deduced mature vde also contains a lipocalinsignature, a domain identified in a number of diverse proteins that bindsmall hydrophobic molecules. For example, crustacyanin, a protein fromlobster carapace which contains a lipocalin signature, binds thecarotenoid astaxanthin. Similarly, this domain may play a role inbinding the substrate violaxanthin. In the third domain approximately47% of the residues have charged side chains. The most striking featureof this domain is the high concentration of glutamic acid residues;27.6% of the residues in this domain (69.2% of the total in the maturevde) are glutamic acids whereas only 2% are aspartic acids.

FIG. 4 provides a detailed analysis of the deduced amino acid sequenceof vde. The top portion provides a comparison of the deduced amino acidsequences of vde from three plant species. The transit peptides arelocated in the boxed region. Identical residues are indicated by hyphens(-). Gaps introduced to maximize sequence alignment are indicated byperiods (.). Asterisks (*) identify the 13 cysteine residues that areconserved between the three sequences.

The bottom map of FIG. 4 shows the three domains identified. The aminoacid spanning regions for lettuce vde are indicated below the domains.

FIG. 6 provides hyropathy profiles for the vdes from three species.

Example 2--Expression of Lettuce vde cDNA in E. coli

Authenticity of the lettuce vde cDNA was confirmed by expression of thefragment vde2 in E. coli. Vde2 was subcloned in both sense and antisenseorientations with respect to lacZ into the Notl site of pGEM-5Zf andtransformed into E. coli DH5alpha. All cultures were incubated andinduced with 10 mM IPTG (Bugos, Plant Mol Biol. (1991)17:1203-1215).Following the 2 hr induction, the cells were centrifuged at 4000 xg for10 min at 4° C. The cells were resuspended in 3 ml 50 mM Tris (pH 7.4),1 mM EDTA and lysed using an ultrasonic cell disrupter equipped with amicro-probe for 10 cycles (30 sec on/30 sec off) while being cooled inan ice bath. The resulting extract was centrifuged at 10,000 xg for 10min at 4° C. and the supernatant was collected for determining vdeactivity using the in vitro assay and absorbance change at 502 nm minus540 nm (Yamamoto, Methods Enzymol. (1985)110:303-312). The pellet waswashed with 3 ml 50 mM Tris (pH 7.4),1 mM EDTA and centrifuged. Thepellet was resuspended in 3 ml buffer and assayed. All assays contained100 μl E. coli extract or pellet resuspension. For quantification ofxanthophyll pigments, the reactions were stopped at various times withaddition of solid Tris and the xanthophylls were extracted 3 times withdiethyl ether. The ether was dried under a stream of N₂ and thexanthophylls were solubilized in 100 μl 90% acetone followed by HPLCanalysis (Gilmore, J. Chromatogr. (1991)543:137-145).

Extracts from E. coli expressing the fragment orientated with lacZ(sense) had strong vde activity whereas no detectable activity wasobserved from extracts of E. coli transformed with vde2 in antisenseorientation or pGEM-5Zf alone. Furthermore, addition of DTT, a stronginhibitor of de-epoxidase activity, abolished all vde activity. DTT(3mM, final conc.) was added directly to the assay 50 seconds afterascorbate (30 mM, final conc.) addition. Specific activity of the enzymewas 64.9±5.4 nmols violaxanthin deepoxidized/min/mg protein. Traceactivity was detected in the membrane fraction of vde2 sense suggestingthat some of the enzyme was not washed away following lysis or thatlysis was not complete. An attempt to express the vde1 fragment wasunsuccessful. E. coli transformed with vde1 subcloned in pGEM-5Zf andorientated with lacZ did not grow.

To verify the products of de-epoxidation, the reaction with vde2 senseextract was stopped at various times and the xanthophylls were analyzedby HPLC. Antheraxanthin and zeaxanthin appeared consistent withsequential de-epoxidation and concomitant with the rapid decrease inviolaxanthin, similar to observations reported over three decadesearlier for de-epoxidation in lima bean (Phaseolus leunatus) leavesexposed to high light (Yamamoto, Arch. Biochem. Biophys.(1962)97:168-173). The specific activity of the enzyme was 19.4±0.9nmols violaxanthin de-epoxidized/min/mg protein. This is the firstunequivocal evidence that the same enzyme catalyzes the two-step monode-epoxidation reaction.

Example 3--vde in Other Plants

Western analysis of vde from chloroplasts of various C₃ plants andexpressed vde in E. coli demonstrate that the N-terminus is conserved.

Intact chloroplasts were isolated (Neubauer, Plant Physiol.(1992)99:1354-1361) and lysed with five freeze/thaw cycles using liquidN₂ (Hager, Planta (1975)88:27-44). Expression of vde2 in E. coliDH5-alpha was as described in Example 2 and the cells were lysed usingthe freeze/thaw method. Proteins were resolved on a 12%SDS-polyacrylamide gel and electrophoretically transferred to PVDF.Color development was performed following incubation with alkalinephosphatase-conjugated secondary antibodies. Protein was estimated usinga prepared reagent (Biorad) and bovine gamma globulin as the standard.

The blot was probed with a polyclonal antibody prepared against asynthetic peptide derived from the N-terminus of lettuce vde(VDALKTCACLLK). Vde migrates with an approximate size of 43 kD.

The mature vde from market romaine lettuce, tobacco (Nicotiana tabacumL. cv Xanthi) and market spinach (Spinacia oleracea L.) all migrate withapproximately the same Mr of 43K. The antibody recognized vde in thesethree plant species demonstrating that the N-terminus is conserved.Expressed vde2 in E. coli migrated at the same M_(r) as the romainelettuce vde whereas extracts from E. coli containing only pGEM-5Zfproduce some minor cross-reacting proteins, none of which having a M_(r)of 43K. The M_(r) 's of the above vde proteins are in agreement with thecalculated M_(r) of the deduced mature vde (39.9K). Two interestingobservations are evident from vde expressed in E. coli. The first isthat the E. coli expressed vde produced many immunoreactive bands oflower molecular weight. Reasons for this may be due to some processingoccurring at the C-terminus of the protein by E. coli (since theantibody recognizes the N-terminus) or due to translational pausing. Thesecond is that the bacterial expressed vde protein migrates at the samemolecular weight as mature vde from romaine lettuce and not as theexpected size of the deduced vde preprotein (54.4K) with the transitpeptide. This suggests that E. coli may recognize the chloroplasttransit peptide and cleave it. The N-terminus of the bacterial expressedvde will need to be sequenced to determine the actual site wherecleavage is occurring. A similar observation was also reported for thenuclear-encoded chloroplast enzyme acetolactate synthase fromArabidopsis when expressed in E. coli.

FIG. 7 shows the kinetics of absorbance change demonstrating expressionof active violaxanthin de-epoxidase in E. coli DH5 (top of FIG. 7).Expression was assayed from vde2 constructs in both sense and antisenseorientations with respect to lacZ along with E. coli containing thevector only (pGEM-5Zf). DTT (3 mM, final concentration) was addeddirectly to the assay 70 seconds after ascorbate (30 mM, finalconcentration) additioin. Specific activity of the enzyme was 64.9±5.4nmols violaxanthin de-epoxidized min -1 mg. protein -1.

The bottom of FIG. 7 is a timecourse of xanthophyll conversions byexpressed vde2 (sense construct) in E. coli. Specific activity of theenzyme was 19.4±0.9 nmols violaxanthin de-epoxidized min -1 protein -1.

Example 4--Effects of Expression of vde in Plants

In FIG. 8, pigment analysis of leaves of 212 control tobacco plants(Ct-#) is provided, as well as the mean percentage of violaxanthin whichis de-epoxidized. Also provided by FIG. 8 is the pigment analysis ofleaves of 18 vde-antisense tobacco plants (TAS-#).

Tobacco plants were transformed with an antisense construct of thetobacco vde cDNA under control of the CaMV 35S promoter (pB1121) usingAgrobacterium tumefaciens LBA4404. A total of 40 antisense plants wereanalyzed with 18 showing various levels of inhibition of de-epoxidation.

Relative pigment concentration for tobacco (Nicotiana tabacum L. cv.Xanthi) leaves was measured by leaf disks punched from tobacco leavesthat were dark adapted for a few hours. One leaf disk (dark adapted) wasextracted with acetone and analyzed by HPLC while another was lightinduced by exposing the disk to 1800 umol m -2 s -1 white light for 20min while the leaf disk was floating on water in a water-jacketed beakercooled at 20° C. Following the light treatment, the leaf disk wasextracted and analyzed by HPLC.

Two vde-antisense tobacco plants (TAS-32 and TAS-39) were recovered thathad undetectable levels of zeaxanthin following illumination with brightwhite light. Low levels of antheraxanthin (˜2-3%) were present in somedark-adapted leaves and are assumed to represent incomplete epoxidaseactivity.

In FIGS. 9 and 10, results are provided from a comparison ofmeasurements on a tobacco leaf from a control plant (Ct-30) and avde-antisense plant (TAS-5), both of which were dark adapted for 24hours. Under low light conditions, three leaf disks were punched fromeach leaf. One leaf disk (dark adapted) was extracted and analyzed byHPLC.

The remaining two leaf disks were pre-illuminated with 500 umol m -2 s-1 red light for 15 minutes. One of these disks was then extracted andanalyzed by HPLC while the other was placed in the dark for 10 minutesprior to fluorometry and HPLC analysis.

It has also been observed that in tobacco plants where lettuce vde hasbeen overexpressed from a 35S construct, flowering is delayed, andflowers are slightly larger.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 3                                             - (2) INFORMATION FOR SEQ ID NO: 1:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:   1981                                                  #acid     (B) TYPE:   nucleic                                                           (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:   line - #ar                                          -     (ii) MOLECULE TYPE: cDNA to mRNA                                        #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - TGTGGGTTCG AATTTTACCC ACCACAAGTT TTGTCCTACC ATAATTGGGA TA - #AGGAGTCT         60                                                                          - AATTTCCCTT GTACAATTTT CCAATTTCTT CCTCCGCCAC ACCATATATA TA - #CTGTACGC        120                                                                          - CACTTCGAAC GCTACAATGT TTGAAAAAAG ACGCAGATTT TACAAAGACG GA - #GAAGATAA        180                                                                          - TAAGCTTCAA GTACTCCGAT CGTCAGGTGG CCTTTGGAAG CCAACAAACT GG - #CT ATG          237                                                                          #      Met                                                                    #        1                                                                    - GCT CTT TCT CTT CAC ACT GTA TTT CTC TGC AA - #A GAG GAA GCC CTC AAT          285                                                                          Ala Leu Ser Leu His Thr Val Phe Leu Cys Ly - #s Glu Glu Ala Leu Asn           #              15                                                             - TTA TAT GCA AGA TCA CCA TGT AAT GAA AGG TT - #T CAC AGG AGT GGA CAA          333                                                                          Leu Tyr Ala Arg Ser Pro Cys Asn Glu Arg Ph - #e His Arg Ser Gly Gln           #         30                                                                  - CCT CCT ACC AAC ATA ATC ATG ATG AAA ATT CG - #A TCC AAC AAT GGA TAT          381                                                                          Pro Pro Thr Asn Ile Ile Met Met Lys Ile Ar - #g Ser Asn Asn Gly Tyr           #     45                                                                      - TTT AAT TCT TTC CGG TTG TTT ACA TCT TAT AA - #G ACA AGT TCT TTC TCA          429                                                                          Phe Asn Ser Phe Arg Leu Phe Thr Ser Tyr Ly - #s Thr Ser Ser Phe Ser           # 65                                                                          - GAT TCT AGC CAT TGC AAG GAT AAA TCT CAG AT - #A TGC AGC ATC GAT ACA          477                                                                          Asp Ser Ser His Cys Lys Asp Lys Ser Gln Il - #e Cys Ser Ile Asp Thr           #                 80                                                          - AGT TTT GAG GAA ATA CAA AGA TTT GAT CTC AA - #A AGG GGC ATG ACT TTG          525                                                                          Ser Phe Glu Glu Ile Gln Arg Phe Asp Leu Ly - #s Arg Gly Met Thr Leu           #             95                                                              - ATT CTT GAA AAG CAA TGG AGA CAA TTC ATA CA - #A TTG GCT ATC GTA TTG          573                                                                          Ile Leu Glu Lys Gln Trp Arg Gln Phe Ile Gl - #n Leu Ala Ile Val Leu           #       110                                                                   - GTT TGC ACA TTT GTT ATC GTT CCC AGA GTT GA - #T GCC GTT GAT GCT CTT          621                                                                          Val Cys Thr Phe Val Ile Val Pro Arg Val As - #p Ala Val Asp Ala Leu           #   125                                                                       - AAA ACT TGT GCT TGT TTA CTC AAA GAA TGC AG - #G ATT GAG CTT GCA AAA          669                                                                          Lys Thr Cys Ala Cys Leu Leu Lys Glu Cys Ar - #g Ile Glu Leu Ala Lys           130                 1 - #35                 1 - #40                 1 -       #45                                                                           - TGT ATA GCA AAC CCA TCT TGT GCG GCA AAC GT - #T GCC TGT CTA CAG ACT          717                                                                          Cys Ile Ala Asn Pro Ser Cys Ala Ala Asn Va - #l Ala Cys Leu Gln Thr           #               160                                                           - TGC AAC AAT CGT CCT GAC GAG ACC GAA TGT CA - #G ATA AAA TGT GGT GAC          765                                                                          Cys Asn Asn Arg Pro Asp Glu Thr Glu Cys Gl - #n Ile Lys Cys Gly Asp           #           175                                                               - TTG TTC GAA AAC AGT GTG GTG GAC CAA TTC AA - #C GAG TGT GCG GTT TCC          813                                                                          Leu Phe Glu Asn Ser Val Val Asp Gln Phe As - #n Glu Cys Ala Val Ser           #       190                                                                   - CGA AAG AAA TGT GTG CCC CGG AAA TCG GAT GT - #G GGT GAA TTC CCG GTT          861                                                                          Arg Lys Lys Cys Val Pro Arg Lys Ser Asp Va - #l Gly Glu Phe Pro Val           #   205                                                                       - CCG GAT CGT AAT GCA GTG GTT CAA AAT TTT AA - #C ATG AAA GAC TTT AGT          909                                                                          Pro Asp Arg Asn Ala Val Val Gln Asn Phe As - #n Met Lys Asp Phe Ser           210                 2 - #15                 2 - #20                 2 -       #25                                                                           - GGG AAG TGG TAT ATA ACA AGT GGT TTA AAT CC - #T ACA TTT GAT GCA TTT          957                                                                          Gly Lys Trp Tyr Ile Thr Ser Gly Leu Asn Pr - #o Thr Phe Asp Ala Phe           #               240                                                           - GAT TGT CAA CTT CAT GAG TTT CAT ATG GAA AA - #T GAT AAA CTT GTT GGG         1005                                                                          Asp Cys Gln Leu His Glu Phe His Met Glu As - #n Asp Lys Leu Val Gly           #           255                                                               - AAC TTA ACA TGG CGC ATA AAA ACT TTG GAT GG - #T GGT TTC TTT ACT CGA         1053                                                                          Asn Leu Thr Trp Arg Ile Lys Thr Leu Asp Gl - #y Gly Phe Phe Thr Arg           #       270                                                                   - TCT GCT GTG CAA ACA TTT GTT CAA GAT CCA GA - #T CTT CCT GGA GCA CTT         1101                                                                          Ser Ala Val Gln Thr Phe Val Gln Asp Pro As - #p Leu Pro Gly Ala Leu           #   285                                                                       - TAT AAT CAT GAC AAT GAG TTT CTT CAC TAC CA - #A GAT GAC TGG TAC ATA         1149                                                                          Tyr Asn His Asp Asn Glu Phe Leu His Tyr Gl - #n Asp Asp Trp Tyr Ile           290                 2 - #95                 3 - #00                 3 -       #05                                                                           - TTA TCT TCC CAA ATC GAA AAC AAA CCC GAT GA - #T TAC ATA TTC GTA TAC         1197                                                                          Leu Ser Ser Gln Ile Glu Asn Lys Pro Asp As - #p Tyr Ile Phe Val Tyr           #               320                                                           - TAC CGA GGT CGA AAC GAC GCA TGG GAT GGA TA - #C GGT GGG TCC GTG ATC         1245                                                                          Tyr Arg Gly Arg Asn Asp Ala Trp Asp Gly Ty - #r Gly Gly Ser Val Ile           #           335                                                               - TAC ACC CGA AGC CCG ACA CTC CCC GAA TCG AT - #C ATC CCA AAC CTA CAA         1293                                                                          Tyr Thr Arg Ser Pro Thr Leu Pro Glu Ser Il - #e Ile Pro Asn Leu Gln           #       350                                                                   - AAA GCA GCC AAA TCC GTG GGT CGA GAC TTT AA - #C AAT TTC ATA ACA ACC         1341                                                                          Lys Ala Ala Lys Ser Val Gly Arg Asp Phe As - #n Asn Phe Ile Thr Thr           #   365                                                                       - GAC AAT AGT TGT GGG CCT GAG CCT CCA TTG GT - #G GAA AGG CTT GAG AAA         1389                                                                          Asp Asn Ser Cys Gly Pro Glu Pro Pro Leu Va - #l Glu Arg Leu Glu Lys           370                 3 - #75                 3 - #80                 3 -       #85                                                                           - ACA GCG GAA GAG GGC GAG AAG TTG TTG ATA AA - #A GAA GCT GTA GAG ATA         1437                                                                          Thr Ala Glu Glu Gly Glu Lys Leu Leu Ile Ly - #s Glu Ala Val Glu Ile           #               400                                                           - GAA GAA GAG GTT GAA AAA GAG GTG GAG AAG GT - #T AGA GAT ACT GAG ATG         1485                                                                          Glu Glu Glu Val Glu Lys Glu Val Glu Lys Va - #l Arg Asp Thr Glu Met           #           415                                                               - ACT TTG TTT CAG AGG TTG CTT GAA GGG TTT AA - #G GAG TTG CAA CAA GAT         1533                                                                          Thr Leu Phe Gln Arg Leu Leu Glu Gly Phe Ly - #s Glu Leu Gln Gln Asp           #       430                                                                   - GAA GAG AAT TTT GTG AGG GAG TTG AGT AAA GA - #A GAG AAG GAA ATT CTG         1581                                                                          Glu Glu Asn Phe Val Arg Glu Leu Ser Lys Gl - #u Glu Lys Glu Ile Leu           #   445                                                                       - AAT GAA CTT CAA ATG GAA GCG ACT GAA GTT GA - #A AAG CTT TTT GGG CGC         1629                                                                          Asn Glu Leu Gln Met Glu Ala Thr Glu Val Gl - #u Lys Leu Phe Gly Arg           450                 4 - #55                 4 - #60                 4 -       #65                                                                           - GCG TTA CCG ATT AGG AAA CTT AGA TAAATTT CGATG - #ATTGA TTCAGACAAT           1680                                                                          Ala Leu Pro Ile Arg Lys Leu Arg                                                               470                                                           - ATATATAGTC ATATGGATTA TGTAGATACT AGAGAAAACC CAAAAAAACT TT - #TGTATACG       1740                                                                          - TGATAAACGT GTTTGTGATT TGTTTATTGG CTTAAAATTG TAGAATAGCT TT - #TTTAATTC       1800                                                                          - TTTACAAAAA AATTGATTGT CTATTGGTAG CCAAGAGGTT CACGAAAAGA CT - #GAAAGGGT       1860                                                                          - CTTGCCGGT TTGCGGGTTA GGCCAAATTT TTTGGGGCGG GATCGGTCTT GAT - #CGGGTTTT       1920                                                                          - TCTTTAAAA CATGTATTTT TTATAAATGA TGAGTTATTT TCAATTTTTG GCT - #AAAAAAAA       1980                                                                          #             1981                                                            - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:   1589                                                  #acid     (B) TYPE:   nucleic                                                           (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:   line - #ar                                          -     (ii) MOLECULE TYPE:   cDNA to mRNA                                      #2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #CTT GCC CCT     56CAGT TGGTGGTAAT ACGGTTGAAG A ATG GCT                       #          Met Ala Leu Ala Pro                                                #         5  1                                                                - CAT TCA AAT TTT CTG GCC AAC CAT GAA ACC AT - #C AAA TAT TAT GTT GGG          104                                                                          His Ser Asn Phe Leu Ala Asn His Glu Thr Il - #e Lys Tyr Tyr Val Gly           #                 20                                                          - TCA AAG CTT CCC GGT CAT AAA AGG TTT AGC TG - #G GGT TGG GAA GAT TAC          152                                                                          Ser Lys Leu Pro Gly His Lys Arg Phe Ser Tr - #p Gly Trp Glu Asp Tyr           #             35                                                              - TTT GGT AGT ATA GTC GTA GCA AAA ATT TGT TC - #C AGC AGA CGG ATA CCT          200                                                                          Phe Gly Ser Ile Val Val Ala Lys Ile Cys Se - #r Ser Arg Arg Ile Pro           #         50                                                                  - AGA TAC TTT CGA AAA TCT CCT AGA ATA TGC TG - #T GGT TTG GAT TCA AGA          248                                                                          Arg Tyr Phe Arg Lys Ser Pro Arg Ile Cys Cy - #s Gly Leu Asp Ser Arg           #     65                                                                      - GGT CTG CAA CTA TTC TCA CAC GGG AAA CAC AA - #T CTC TCT CCC GCA CAT          296                                                                          Gly Leu Gln Leu Phe Ser His Gly Lys His As - #n Leu Ser Pro Ala His           # 85                                                                          - AGC ATT AAC CAG AAT GTA CCT AAG GGA AAT TC - #A GGA TGC AAA TTT CCA          344                                                                          Ser Ile Asn Gln Asn Val Pro Lys Gly Asn Se - #r Gly Cys Lys Phe Pro           #                100                                                          - AAA GAT GTA GCT TTG ATG GTT TGG GAG AAA TG - #G GGC CAA TTT GCC AAA          392                                                                          Lys Asp Val Ala Leu Met Val Trp Glu Lys Tr - #p Gly Gln Phe Ala Lys           #           115                                                               - ACA GCA ATT GTA GCT ATA TTC ATT TTG TCA GT - #T GCT TCA AAA GCT GAT          440                                                                          Thr Ala Ile Val Ala Ile Phe Ile Leu Ser Va - #l Ala Ser Lys Ala Asp           #       130                                                                   - GCG GTT GAT GCT CTC AAG ACT TGT ACT TGC TT - #A CTG AAA GAG TGC AGG          488                                                                          Ala Val Asp Ala Leu Lys Thr Cys Thr Cys Le - #u Leu Lys Glu Cys Arg           #   145                                                                       - TTA GAG CTT GCG AAG TGC ATT TCG AAC CCT GC - #A TGT GCA GCT AAT GTT          536                                                                          Leu Glu Leu Ala Lys Cys Ile Ser Asn Pro Al - #a Cys Ala Ala Asn Val           150                 1 - #55                 1 - #60                 1 -       #65                                                                           - GCC TGT CTC CAG ACT TGC AAC AAT AGA CCT GA - #C GAA ACG GAA TGT CAG          584                                                                          Ala Cys Leu Gln Thr Cys Asn Asn Arg Pro As - #p Glu Thr Glu Cys Gln           #               180                                                           - ATA AAA TGT GGT GAT TTG TTT GAA AAC AGT GT - #C GTA GAC GAG TTC AAT          632                                                                          Ile Lys Cys Gly Asp Leu Phe Glu Asn Ser Va - #l Val Asp Glu Phe Asn           #           195                                                               - GAG TGT GCA GTC TCC CGA AAG AAA TGT GTA CC - #T CGT AAA TCT GAT GTT          680                                                                          Glu Cys Ala Val Ser Arg Lys Lys Cys Val Pr - #o Arg Lys Ser Asp Val           #       210                                                                   - GGT GAC TTT CCT GTA CCT GAT CCC AGT GTT CT - #T GTC CAG AAG TTT GAC          728                                                                          Gly Asp Phe Pro Val Pro Asp Pro Ser Val Le - #u Val Gln Lys Phe Asp           #   225                                                                       - ATG AAA GAT TTT AGC GGG AAA TGG TTC ATT AC - #T CGC GGT TTG AAT CCC          776                                                                          Met Lys Asp Phe Ser Gly Lys Trp Phe Ile Th - #r Arg Gly Leu Asn Pro           230                 2 - #35                 2 - #40                 2 -       #45                                                                           - ACT TTT GAT GCT TTT GAT TGC CAA TTG CAT GA - #G TTC CAT ACA GAA GAA          824                                                                          Thr Phe Asp Ala Phe Asp Cys Gln Leu His Gl - #u Phe His Thr Glu Glu           #               260                                                           - AAC AAA CTT GTG GGG AAT TTA TCT TGG AGA AT - #A CGT ACA CCT GAT GGA          872                                                                          Asn Lys Leu Val Gly Asn Leu Ser Trp Arg Il - #e Arg Thr Pro Asp Gly           #           275                                                               - GGA TTT TTT ACT CGA TCA GCG GTG CAA AAA TT - #C GTG CAA GAT CCA AAG          920                                                                          Gly Phe Phe Thr Arg Ser Ala Val Gln Lys Ph - #e Val Gln Asp Pro Lys           #       290                                                                   - TAT CCG GGG ATA CTC TAC AAT CAT GAT AAT GA - #G TAT CTT CTC TAC CAA          968                                                                          Tyr Pro Gly Ile Leu Tyr Asn His Asp Asn Gl - #u Tyr Leu Leu Tyr Gln           #   305                                                                       - GAT GAC TGG TAT ATT TTG TCA TCC AAA GTA GA - #A AAT AGT CCA GAG GAT         1016                                                                          Asp Asp Trp Tyr Ile Leu Ser Ser Lys Val Gl - #u Asn Ser Pro Glu Asp           310                 3 - #15                 3 - #20                 3 -       #25                                                                           - TAC ATA TTT GTG TAC TAT AAG GGC AGA AAT GA - #T GCA TGG GAT GGA TAT         1064                                                                          Tyr Ile Phe Val Tyr Tyr Lys Gly Arg Asn As - #p Ala Trp Asp Gly Tyr           #               340                                                           - GGT GGT TCT GTA CTT TAC ACA AGA AGT GCA GT - #T TTG CCT GAA AGC ATT         1112                                                                          Gly Gly Ser Val Leu Tyr Thr Arg Ser Ala Va - #l Leu Pro Glu Ser Ile           #           355                                                               - ATA CCG GAG TTG CAA ACC GCA GCT CAA AAA GT - #T GGG CGT GAT TTC AAC         1160                                                                          Ile Pro Glu Leu Gln Thr Ala Ala Gln Lys Va - #l Gly Arg Asp Phe Asn           #       370                                                                   - ACA TTC ATA AAA ACA GAC AAT ACA TGT GGC CC - #T GAA CCT CCC CTT GTT         1208                                                                          Thr Phe Ile Lys Thr Asp Asn Thr Cys Gly Pr - #o Glu Pro Pro Leu Val           #   385                                                                       - GAG AGG TTG GAG AAG AAA GTG GAA GAA GGA GA - #A AGG ACG ATC ATA AAA         1256                                                                          Glu Arg Leu Glu Lys Lys Val Glu Glu Gly Gl - #u Arg Thr Ile Ile Lys           390                 3 - #95                 4 - #00                 4 -       #05                                                                           - GAA GTT GAG GAG ATA GAA GAA GAA GTA GAG AA - #G GTG AGA GAT AAA GAA         1304                                                                          Glu Val Glu Glu Ile Glu Glu Glu Val Glu Ly - #s Val Arg Asp Lys Glu           #               420                                                           - GTC ACC TTA TTC AGT AAA CTG TTT GAA GGT TT - #T AAA GAG CTC CAA CGA         1352                                                                          Val Thr Leu Phe Ser Lys Leu Phe Glu Gly Ph - #e Lys Glu Leu Gln Arg           #           435                                                               - GAT GAA GAG AAC TTC TTA AGA GAG CTG AGC AA - #A GAA GAA ATG GAT GTT         1400                                                                          Asp Glu Glu Asn Phe Leu Arg Glu Leu Ser Ly - #s Glu Glu Met Asp Val           #       450                                                                   - TTG GAT GGA CTT AAA ATG GAA GCA ACT GAG GT - #A GAA AAA CTT TTT GGG         1448                                                                          Leu Asp Gly Leu Lys Met Glu Ala Thr Glu Va - #l Glu Lys Leu Phe Gly           #   465                                                                       #ATTTTTAAAA CTATCAACAT     1500TA A GGTAAGT                                   Arg Ala Leu Pro Ile Arg Lys Leu                                               470                 4 - #75                                                   - ATATACTACA TGTATAGTTG TATTTGATTC TTTTGCCTGG AATAGATTGC TT - #ATACATCA       1560                                                                          #          1589    CAGA AGCAAAAAA                                             - (2) INFORMATION FOR SEQ ID NO: 3:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:   1555                                                  #acid     (B) TYPE:   nucleic                                                           (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:   line - #ar                                          -     (ii) MOLECULE TYPE:   cDNA to mRNA                                      #3:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - CCACGCGTCC GGCTTGGTGT GGGGAAGATT AGATAGTGTG AAGA ATG G - #CA GTA GCT          56                                                                          #Ala          Met Ala Val                                                     #               1                                                             - ACA CAT TGT TTC ACT TCA CCT TGT CAT GAC CG - #T ATT CGA TTT TTC TCA          104                                                                          Thr His Cys Phe Thr Ser Pro Cys His Asp Ar - #g Ile Arg Phe Phe Ser           #  20                                                                         - AGT GAT GAT GGT ATT GGT AGG CTT GGC ATT AC - #A AGA AAG AGG ATC AAT          152                                                                          Ser Asp Asp Gly Ile Gly Arg Leu Gly Ile Th - #r Arg Lys Arg Ile Asn           #                 35                                                          - GGC ACT TTC TTG CTC AAG ATT TTA CCT CCA AT - #C CAA AGT GCT GAT CTC          200                                                                          Gly Thr Phe Leu Leu Lys Ile Leu Pro Pro Il - #e Gln Ser Ala Asp Leu           #             50                                                              - AGA ACA ACT GGT GGG AGA TCC TCA CGT CCT TT - #A TCT GCA TTC AGG TCA          248                                                                          Arg Thr Thr Gly Gly Arg Ser Ser Arg Pro Le - #u Ser Ala Phe Arg Ser           #         65                                                                  - GGA TTC TCT AAG GGG ATA TTT GAC ATT GTG CC - #A TTA CCA TCA AAG AAT          296                                                                          Gly Phe Ser Lys Gly Ile Phe Asp Ile Val Pr - #o Leu Pro Ser Lys Asn           #     80                                                                      - GAG CTG AAA GAG CTG ACC GCT CCG CTG TTG CT - #A AAA CTC GTG GGT GTT          344                                                                          Glu Leu Lys Glu Leu Thr Ala Pro Leu Leu Le - #u Lys Leu Val Gly Val           #100                                                                          - TTA GCT TGC GCG TTC CTT ATT GTT CCA TCT GC - #A GAT GCA GTT GAT GCA          392                                                                          Leu Ala Cys Ala Phe Leu Ile Val Pro Ser Al - #a Asp Ala Val Asp Ala           #               115                                                           - CTT AAA ACT TGT GCA TGC TTA TTG AAG GGA TG - #C AGG ATA GAA CTC GCA          440                                                                          Leu Lys Thr Cys Ala Cys Leu Leu Lys Gly Cy - #s Arg Ile Glu Leu Ala           #           130                                                               - AAG TGC ATT GCC AAC CCT GCC TGT GCA GCC AA - #T GTC GCG TGC CTT CAG          488                                                                          Lys Cys Ile Ala Asn Pro Ala Cys Ala Ala As - #n Val Ala Cys Leu Gln           #       145                                                                   - ACC TGC AAT AAC CGT CCA GAT GAA ACC GAG TG - #C CAG ATT AAA TGT GGG          536                                                                          Thr Cys Asn Asn Arg Pro Asp Glu Thr Glu Cy - #s Gln Ile Lys Cys Gly           #   160                                                                       - GAT CTG TTT GAG AAC AGT GTT GTT GAT GAG TT - #C AAC GAG TGT GCT GTG          584                                                                          Asp Leu Phe Glu Asn Ser Val Val Asp Glu Ph - #e Asn Glu Cys Ala Val           165                 1 - #70                 1 - #75                 1 -       #80                                                                           - TCG AGA AAA AAG TGT GTT CCT AGA AAA TCT GA - #T CTC GGA GAA TTT CCT          632                                                                          Ser Arg Lys Lys Cys Val Pro Arg Lys Ser As - #p Leu Gly Glu Phe Pro           #               195                                                           - GCC CCA GAC CCT TCT GTT CTT GTA CAG AAC TT - #C AAC ATC TCG GAC TTT          680                                                                          Ala Pro Asp Pro Ser Val Leu Val Gln Asn Ph - #e Asn Ile Ser Asp Phe           #           210                                                               - AAC GGG AAG TGG TAC ATT ACA AGT GGC TTG AA - #T CCA ACC TTT GAT GCC          728                                                                          Asn Gly Lys Trp Tyr Ile Thr Ser Gly Leu As - #n Pro Thr Phe Asp Ala           #       225                                                                   - TTC GAC TGC CAG CTG CAT GAG TTC CAC ACA GA - #A GGT GAC AAC AAG CTT          776                                                                          Phe Asp Cys Gln Leu His Glu Phe His Thr Gl - #u Gly Asp Asn Lys Leu           #   240                                                                       - GTT GGA AAC ATC TCT TGG AGA ATA AAG ACC CT - #A GAC AGT GGA TTC TTT          824                                                                          Val Gly Asn Ile Ser Trp Arg Ile Lys Thr Le - #u Asp Ser Gly Phe Phe           245                 2 - #50                 2 - #55                 2 -       #60                                                                           - ACT AGG TCA GCC GTA CAA AAA TTC GTG CAA GA - #T CCT AAC CAA CCT GGT          872                                                                          Thr Arg Ser Ala Val Gln Lys Phe Val Gln As - #p Pro Asn Gln Pro Gly           #               275                                                           - GTT CTC TAC AAT CAT GAC AAC GAG TAC CTT CA - #C TAT CAA GAT GAC TGG          920                                                                          Val Leu Tyr Asn His Asp Asn Glu Tyr Leu Hi - #s Tyr Gln Asp Asp Trp           #           290                                                               - TAT ATC CTG TCA TCA AAG ATA GAG AAT AAA CC - #T GAA GAC TAT ATA TTT          968                                                                          Tyr Ile Leu Ser Ser Lys Ile Glu Asn Lys Pr - #o Glu Asp Tyr Ile Phe           #       305                                                                   - GTA TAC TAC CGT GGG CGA AAC GAT GCT TGG GA - #T GGA TAT GGT GGT GCA         1016                                                                          Val Tyr Tyr Arg Gly Arg Asn Asp Ala Trp As - #p Gly Tyr Gly Gly Ala           #   320                                                                       - GTT GTA TAC ACG AGA AGT TCT GTA TTA CCC AA - #T AGC ATT ATA CCA GAA         1064                                                                          Val Val Tyr Thr Arg Ser Ser Val Leu Pro As - #n Ser Ile Ile Pro Glu           325                 3 - #30                 3 - #35                 3 -       #40                                                                           - CTC GAA AAA GCA GCA AAA AGC ATA GGC AGA GA - #C TTC AGC ACA TTC ATT         1112                                                                          Leu Glu Lys Ala Ala Lys Ser Ile Gly Arg As - #p Phe Ser Thr Phe Ile           #               355                                                           - AGA ACG GAT AAC ACA TGT GGT CCT GAA CCT GC - #G CTC GTG GAG AGA ATT         1160                                                                          Arg Thr Asp Asn Thr Cys Gly Pro Glu Pro Al - #a Leu Val Glu Arg Ile           #           370                                                               - GAG AAG ACA GTG GAA GAA GGT GAA AGG ATA AT - #C GTA AAA GAG GTT GAA         1208                                                                          Glu Lys Thr Val Glu Glu Gly Glu Arg Ile Il - #e Val Lys Glu Val Glu           #       385                                                                   - GAG ATA GAA GAA GAG GTA GAG AAG GAA GTG GA - #G AAG GTC GGT AGG ACT         1256                                                                          Glu Ile Glu Glu Glu Val Glu Lys Glu Val Gl - #u Lys Val Gly Arg Thr           #   400                                                                       - GAG ATG ACC TTG TTC CAG AGA TTG GCT GAA GG - #A TTT AAT GAA CTG AAG         1304                                                                          Glu Met Thr Leu Phe Gln Arg Leu Ala Glu Gl - #y Phe Asn Glu Leu Lys           405                 4 - #10                 4 - #15                 4 -       #20                                                                           - CAA GAC GAG GAG AAT TTC GTG AGA GAG TTA AG - #T AAA GAA GAG ATG GAG         1352                                                                          Gln Asp Glu Glu Asn Phe Val Arg Glu Leu Se - #r Lys Glu Glu Met Glu           #               435                                                           - TTT TTG GAT GAG ATC AAA ATG GAA GCA AGT GA - #G GTT GAA AAA TTG TTT         1400                                                                          Phe Leu Asp Glu Ile Lys Met Glu Ala Ser Gl - #u Val Glu Lys Leu Phe           #           450                                                               - GGG AAA GCT TTG CCA ATC AGG AAG GTC AGG TA - #GAAACAAG AACCACCATT           1450                                                                          Gly Lys Ala Leu Pro Ile Arg Lys Val Arg                                       #       460                                                                   - GTTGTACAAA CTATATTATA CATACTGTGT TCGGTTCATA TAAAGTAATA TT - #TTTGTACA       1510                                                                          #                1555AA CAATTGGATA AAAAAAAAAA AAAAA                           __________________________________________________________________________

What is claimed is:
 1. An isolated DNA sequence encoding plantviolaxanthin de-epoxidase.
 2. The DNA sequence of claim 1 wherein saidviolaxanthin de-epoxidase DNA sequence is joined to a heterologousnucleic acid sequence.
 3. A recombinant DNA construct capable ofdirecting the transciption of RNA in a plant cell, wherein saidconstruct comprises in the order of transcription, a plant transcriptioninitiation region, the violaxanthin de-epoxidase encoding sequence ofclaim 1, and a transcriptional termination region.
 4. The DNA sequenceof claim 1, wherein said sequence is selected from the group consistingof the nucleic acid sequences in SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO3.
 5. The DNA sequence of claims 1, wherein said sequence encodes atleast about the twenty N-terminus amino acids of a protein selected fromthe group consisting of the plant violaxanthin de-epoxidase proteins inSEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO
 3. 6. The DNA sequence of claim5, wherein said sequence encodes a plant violaxanthin de-epoxidaseprotein selected from the group consisting of the proteins in SEQ ID NO.1, SEQ ID NO 2, and SEQ ID NO
 3. 7. The DNA sequence of claim 1, whereinsaid sequence encodes a protein comprising the amino acids VDALKTCACLLK.8. A method of modifying the violaxanthin de-epoxidase levels in aplant, said method comprising growing a plant transformed by a constructaccording to claim
 3. 9. The method of claim 8 wherein said encodingsequence is in sense orientation.
 10. The method of claim 9 wherein saidconstruct further comprises a plastid translocation sequence.
 11. Amethod of modifying the sensitivity of a transgenic plant to lightcomprising growing a plant transformed by a construct according to claim3.
 12. The method of claim 10 wherein violaxanthin de-epoxidase activityis increased resulting in increased zeaxanthin and antheraxanthinproduction.
 13. The method of claim 12 wherein said increased zeaxanthinand antheraxanthin levels results in said plant being tolerant ofincreased light levels, as opposed to a non-transformed control plant ofthe same type.
 14. A transgenic plant with modified sensitivity to lightas a consequence of the activity of an introduced construct according toclaim
 3. 15. A plant, plant cell or other plant part, each comprising aconstruct according to claim
 3. 16. A plant, plant cell or other plantpart, each produced by the method of claim
 8. 17. A plant, plant cell orother plant part, each produced by the method of claim 10 whereinflowering of said plant is delayed as compared to flowering in a controlplant not produced by said method.
 18. A plant, plant cell or otherplant part, each produced by the method of claim 10 wherein flowers ofsaid plant are larger as compared to flowers of a control plant notproduced by said method.